International Journal of
Molecular Sciences Article
Ghrelin Attenuates Intestinal Barrier Dysfunction Following Intracerebral Hemorrhage in Mice Yijun Cheng 1,† , Yongxu Wei 1,† , Wenlei Yang 1 , Yu Cai 1 , Bin Chen 1 , Guoyuan Yang 2,3 , Hanbing Shang 1 and Weiguo Zhao 1, * 1
2 3
* †
Department of Neurosurgery, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China;
[email protected] (Y.C.);
[email protected] (Y.W.);
[email protected] (W.Y.);
[email protected] (Y.C.);
[email protected] (B.C.);
[email protected] (H.S.) Department of Neurology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China;
[email protected] Neuroscience and Neuroengineering Research Center, Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, China Correspondence:
[email protected]; Tel.: +86-21-6437-0045 (ext. 666092); Fax: +86-21-6433-3548 These authors contributed equally to this work.
Academic Editor: Suzanne L. Dickson Received: 21 September 2016; Accepted: 28 November 2016; Published: 6 December 2016
Abstract: Intestinal barrier dysfunction remains a critical problem in patients with intracerebral hemorrhage (ICH) and is associated with poor prognosis. Ghrelin, a brain-gut peptide, has been shown to exert protection in animal models of gastrointestinal injury. However, the effect of ghrelin on intestinal barrier dysfunction post-ICH and its possible underlying mechanisms are still unknown. This study was designed to investigate whether ghrelin administration attenuates intestinal barrier dysfunction in experimental ICH using an intrastriatal autologous blood infusion mouse model. Our data showed that treatment with ghrelin markedly attenuated intestinal mucosal injury at both histomorphometric and ultrastructural levels post-ICH. Ghrelin reduced ICH-induced intestinal permeability according to fluorescein isothiocyanate conjugated-dextran (FITC-D) and Evans blue extravasation assays. Concomitantly, the intestinal tight junction-related protein markers, Zonula occludens-1 (ZO-1) and claudin-5 were upregulated by ghrelin post-ICH. Additionally, ghrelin reduced intestinal intercellular adhesion molecule-1 (ICAM-1) expression at the mRNA and protein levels following ICH. Furthermore, ghrelin suppressed the translocation of intestinal endotoxin post-ICH. These changes were accompanied by improved survival rates and an attenuation of body weight loss post-ICH. In conclusion, our results suggest that ghrelin reduced intestinal barrier dysfunction, thereby reducing mortality and weight loss, indicating that ghrelin is a potential therapeutic agent in ICH-induced intestinal barrier dysfunction therapy. Keywords: intracerebral hemorrhage; ghrelin; intestinal barrier dysfunction; mucosa; intestinal permeability; tight junction; ICAM-1
1. Introduction Intracerebral hemorrhage (ICH) is the most devastating subtype of stroke and is associated with high morbidity and mortality. This hemorrhagic disorder accounts for 10%–15% of all strokes, with approximately 2 million cases per year worldwide [1]. Importantly, ICH may not only cause primary damage to the brain itself but also lead to secondary damage to remote organs, such as to the gastrointestinal tract, lung, and heart. Notably, intestinal barrier dysfunction is a common complication after ICH that leads to malabsorption, malnutrition, hypoimmunity, and poor prognosis in patients [2,3]. Int. J. Mol. Sci. 2016, 17, 2032; doi:10.3390/ijms17122032
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2016, 17, 2032
2 of 14
Intestinal barrier dysfunction is mainly characterized by mucosal injury and increased intestinal permeability [4,5]. The intestines are the core and initiating organs of multiple organ dysfunction under various stressed conditions. The increased permeability of injured intestinal mucosa could result in the translocation of intestinal endotoxin, which, in turn, triggers both a systemic inflammatory response syndrome (SIRS) and a multiple organ dysfunction syndrome (MODS). In the clinic, patients with ICH manifesting gastrointestinal dysfunction have a longer hospitalization period and a higher mortality [2,6]. Although very common, effective strategies for preventing intestinal barrier dysfunction following ICH are still lacking [7]. Ghrelin, a 28-amino-acid brain-gut peptide mainly secreted from the stomach, acts as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) [8,9]. Ghrelin has numerous biological actions in physiological processes, such as roles in feeding regulation, growth hormone secretion, gastric acid secretion, and metabolism [9,10]. Previous studies have demonstrated that ghrelin can confer protection against intestinal dysfunction in animal models of traumatic brain injury (TBI) [4,5], and sepsis [11]. Nevertheless, until now, no study has addressed the potential effect of ghrelin on ICH-induced intestinal barrier dysfunction. Our study was conducted to test the hypothesis that ghrelin administration attenuates intestinal barrier impairment following ICH in mice. 2. Results 2.1. Ghrelin Improved Histological Changes in the Intestinal Mucosa after ICH To investigate the effect of ghrelin administration on the intestinal mucosa post-ICH, ileum sections from each group were observed at histomorphometric and ultrastructural levels. At the histomorphometric level, the samples showed nearly intact mucosa in the sham group (Figure 1A). However, mucosal damage occurred one day post-ICH, indicated by severe lifting of epithelial cells, thickened and shortened villi, fusion of adjacent villi, and naked lamina propria (Figure 1B). After ghrelin administration, the mucosa was protected from injury, presenting less lifting of epithelial cells, thinner and longer villi, less fusion of adjacent villi and fewer naked lamina propria (Figure 1C). Int. J. Mol. Sci. 2016, 17, 2032 3 of 14
Figure 1. Ghrelin attenuated intestinal mucosal injury after intracerebral hemorrhage (ICH).
Figure 1. Ghrelin attenuated intestinal mucosal injury after intracerebral hemorrhage (ICH). (A) The (A) The sham group showed nearly intact mucosa at the histomorphometric level. Scale bar = 100 µm; sham group (B) showed intact severe mucosa the histomorphometric level. villi, Scale The ICH nearly group presented liftingat of epithelial cells, thickened and shortened fusionbar of = 100 µm; adjacent presented villi, and naked laminalifting propria at histomorphometric level; (C) Lessand lifting of epithelial villi, fusion (B) The ICH group severe ofthe epithelial cells, thickened shortened cells, thinner and longer villi, less fusion of adjacent villi and naked lamina propria were observed in of adjacent villi, and naked lamina propria at the histomorphometric level; (C) Less lifting of the ghrelin-treated group at the histomorphometric level; (D) The sham group showed smooth-surfaced, epithelial cells, thinner and lessat the fusion of adjacent villibarand propria orderly arranged villi longer with few villi, secretions ultrastructural level. Scale = 300naked µm; (E) lamina ICH induced marked ultrastructural group changes,atincluding increased secretions, irregularly arranged, were observed in the ghrelin-treated the histomorphometric level; (D) The sham group thickened, sparse, and surface-ruptured villi; and (F) Less secretions and more intact villi were showed smooth-surfaced, orderly arranged villi with few secretions at the ultrastructural level. observed in the ICH + ghrelin group at the ultrastructural level. n = 4 per group. Scale bar = 300 µm; (E) ICH induced marked ultrastructural changes, including increased secretions, irregularly arranged, thickened, sparse, and surface-ruptured villi; and (F) Less secretions and more intact villi were observed in the ICH + ghrelin group at the ultrastructural level. n = 4 per group.
Int. J. Mol. Sci. 2016, 17, 2032
3 of 14
Int. J. Mol. Sci. 2016, 17, 2032
3 of 14
At the ultrastructural level, sections from the sham group showed smooth-surfaced, orderly arranged villi with few secretions (Figure 1D). Following ICH induction, marked ultrastructural changes were observed, including increased secretions, irregularly arranged, thickened, sparse, and surface-ruptured villi (Figure 1E). After treatment with ghrelin, less secretions and more intact villi were observed (Figure 1F). 2.2. Ghrelin Attenuated Intestinal Permeability after ICH To evaluate the effect of ghrelin on intestinal permeability post-ICH, fluorescein isothiocyanate Figure 1. Ghrelin attenuated intestinal injury after intracerebral (ICH). conjugated-dextran (FITC-D) and Evans bluemucosal extravasation assayshemorrhage were performed. As shown in (A) The sham group showed nearly intact mucosa at the histomorphometric level. Scale bar = 100 µm; Figure 2A, low FITC-D level was detected in the sham ICH caused a significant increase in (B) The ICH group presented severe lifting of epithelial cells,group. thickened and shortened villi, fusion of adjacent villi, and naked lamina propria at the histomorphometric level; (C) Less lifting of epithelial the level of serum FITC-D compared to the sham group (p < 0.05). However, ghrelin administration cells, thinner and longer villi, less fusion of adjacent villi and naked lamina propria were observed in ghrelin-treated grouppost-ICH at the histomorphometric level; (D) The sham group smooth-surfaced, markedly decreased the FITC-D level (p < 0.05). Based on showed Evans blue extravasation, the ICH orderly arranged villi with few secretions at the ultrastructural level. Scale bar = 300 µm; (E) ICH group exhibited higher dye extravasation compared to the sham group (p 0.05). However, treatment induced marked ultrastructural changes, including increased secretions, irregularly
